In recent years, mass spectrometers have been indispensable for identification and structural analysis of a biological sample containing proteins, sugar chains, lipids or the like. In particular, the technique of MSn analysis is extremely useful for the structural of proteins, sugar chains or the like. In an MSn analysis, an ion originating from a sample and having a comparatively large molecular weight is dissociated one or more times by collision induced dissociation (CID) and subjected to mass spectrometry.
A well-known technique for analyzing proteins (or peptides) using a mass spectrometer is a database search method, in which mass-to-charge ratios of the peaks on an MSn spectrum obtained by an MSn analysis of a sample are compared with those of the product ions calculated from proteins registered in a database, and the amino acid sequence of a peptide corresponding to the actual measurement data is deduced based on the degree of matching of the mass-to-charge ratios. The “Mascot MS/MS ion search”, an online system offered by Matrix Science Ltd, UK, is one of the current and most widely used database search techniques (see Non-Patent Document 1).
On the other hand, as a technique for analyzing a sugar-chain structure using a mass spectrometer, a library search is commonly known, in which an MSn spectrum pattern obtained by an MSn analysis of an unknown sample is compared with each of the MSn spectra stored in a spectrum library in which MSn spectra obtained by MSn analyses of samples having known structures are registered, and the sugar-chain structure corresponding to the measurement data is deduced based on the degree of matching of the spectrums (see Non-Patent Document 2). This library search for sugar chain structural analysis differs from the previously described MS/MS ion search aimed at identification of proteins and peptides in that it uses a spectrum library including MSn spectra of sugar chains having known structures in place of a database including structural information of proteins and other kinds of information, although the spectrum library is also a database in the broad sense.
That is to say, although a difference exists in that the target is an amino acid sequence in case of the MS/MS ion search and a sugar-chain structure in the case of the sugar-chain library search, both methods can be categorized as a technique for identifying a sample by using a database. In these techniques, a substance contained in a sample is identified by comparing an actually measured mass spectrum to be identified with theoretical fragment information of peptides obtained from a database or mass spectra obtained by actual measurements of sugar chains. The eventually presented result is selected based on the degree of matching evaluated in the process of comparison.
The reason why the MS/MS ion search is effective for the identification and structural analysis of peptides is because, even if there are different kinds of peptides whose amino acid sequences partially include the same sequence pattern, the theoretical calculation of the mass-to-charge ratios of the product ions will, in many cases, reveal a unique combination of mass-to-charge ratio values of the product ions for each peptide. However, the MS/MS ion search or similar analytical techniques are not very effective for sugar-chain structural analysis. This is because sugar chains are extremely varied in structure and can take a variety of forms with the same mass and composition. Sugar chains having different structures and yet being identical in composition generate a number of product ions with the same combination of mass-to-charge ratios. Therefore, it is impossible to determine the structure of a sugar chain by merely evaluating the degree of matching of the mass-to-charge ratios of the product ions. This is the reason the technique of comparing MSn spectra by library search is used in the sugar-chain analysis.
In a commonly used, conventional sugar-chain library search, the sugar-chain structure of an unknown sample is deduced by comparing an MSn spectrum of the sample with MSn spectra registered in a spectrum library in terms of not only the values of the mass-to-charge ratios of the peaks on the MSn spectra but also the signal intensity information contained in the MSn spectra. Comparing not only the mass-to-charge-ratio values of the peaks on MSn spectra but also their signal intensity values makes it possible to additionally identify structural isomers, such as positional or anomeric isomers. For example, Non-Patent Document 2 and Patent Document 1 suggest that the use of signal intensity information contained in MSn spectra makes it possible to identify a positional isomer in terms of not only which of the carbons in a simple sugar is engaged in the bonding but also whether the bond is an α or β bond.
However, in such a sugar-chain library search, if an MSn spectrum matching the unknown sugar-chain structure of a target sample is not included in the spectrum library, the MSn spectrum obtained by an actual measurement of the target sample cannot be identified at all or is incorrectly identified as a different kind of sugar chain or structure. To improve identification accuracy, it is necessary to enrich the spectrum library, which is an extremely laborious task and requires an enormous amount of time and labor.
Applying the sugar-chain library search requires satisfying the rigorous constraint that the measurement of unknown samples be performed under the same conditions (such as the sample preparation method, labeling method, and dissociation energy) as used when the MSn spectra included in the spectrum library were obtained. For example, the library search disclosed in Non-Patent Document 2 requires 2-aminopyridine (PA) labeling a sugar-chain sample and performing a measurement of the sample with a matrix-assisted laser desorption/ionization ion-trap time-of-flight mass spectrometer (MALDI-QIT-TOFMS). The kind of matrix to be used for preparing the sample and the amount of dissociation energy are also limited.